Quantitative and specific detection of biomolecules such as DNA and proteins have a wide variety of applications, for example, in biomedical diagnostics and environmental monitoring. DNA sensing has broad application, for example, in genotyping, gene-expression studies, mutation detection, pharmacogenomics, forensics, and related fields in which genetic contents provide insight into biological function or identity. Multiplexed DNA analysis can be performed in a laboratory environment using a “microarray”, a passive substrate (such as a glass slide) on which thousands of single-stranded DNA (ssDNA) “probe” molecules arranged in a regular pattern bind to (or “hybridize” with) fluorophore-labeled “target” molecules in an analyte solution. Probes can be synthesized externally and then immobilized on the microarray through mechanical contact spotting or non-contact ink-jet printing, or can be constructed in situ using photolithographic techniques and solid-phase chemical synthesis. Hybridization occurs, for example, when the probe and target sequences are complementary to one another. Microarray scanners, employing laser sources that excite the fluorophores and photomultiplier tubes or CCD cameras that detect the emitted light, can measure surface-bound target densities down to 106 cm−2. Relative expression levels of bound targets at different array sites can then be quantified from the resulting image. However, fluorescent techniques typically require labeled targets and bulky instrumentation, making them ill-suited for point-of-care applications.
Electrochemical sensing approaches to DNA detection rely on detecting changes occurring with hybridization at the interface between a metal “working” electrode (“WE”), functionalized with probe molecules, and a conductive target analyte solution. One example feedback circuit known as a “potentiostat” can be used to apply a desired potential across the WE interface and measure the resulting current. If the target molecules are conjugated, for example, with “redox” labels (e.g., chemical species which gain electrons (undergo reduction) or lose electrons (undergo oxidation) due to an applied potential), probe-target binding can be detected, for example, by measuring changes in the direct (Faradaic) current flowing across the interface. Alternatively, label-free sensing can be performed, for example, by measuring changes in displacement (non-Faradaic) current at the interface that occur due to surface-charge fluctuations.
Microarray applications based on electrochemical sensing often require parallel detection of hundreds to thousands of sensing sites. This requires active multiplexing that can be achieved through integration of the WEs onto an active complementary metal-oxide-semiconductor (“CMOS”) substrate containing the sensor electronics.
FIGS. 24a-c depict several sensor chip architectures. FIG. 24a depicts a sensor chip interfaced with one off-chip working electrode. FIG. 24b depicts an array of on-chip sensing elements connected individually to off-chip working electrodes. FIG. 24c depicts the integration of arrays of sensing elements with on-chip working electrodes, where all electrochemical reactions are carried out on the sensor chip surface.
A need exists for a technique which supports generalized potentiostat functionality and real-time monitoring of hybridization with the ability to directly measure surface target coverages.